1. Field of the invention
This invention is in the field of assay of encephalopathies in humans and animals. The term "encephalopathy" is used herein to refer to neurodegenerative diseases implicating prions such as scrapie, in sheep and goats, bovine spongiform encephalopathy (BSE) in cattle, mink and cats and Creutzfeld-Jakob Disease (CJD), Gerstmann-Stra ussler-Scheinker Syndrome (GSS) and kuru in humans. The term "assay" is used herein to cover mere detection (the likely ordinary use envisaged) and a quantitative or semi-quantitative procedure in which some estimate is made of the amount of the DNA present in the sample.
2. Description of the related art
The state of knowledge of the causative agent of encaphalopathies has been summarised recently by N. Hunter, TINS 14 (9), 389-390 (1991) and by S. B. Prusiner, M. Torcha and D. Nestaway, Cornell Veterinarian 81 (2), 85-101 (1991).
The infectious agent is a particle which differs from a virion and has been termed a "prion". Prions are thought to contain little or no nucleic acid and are composed largely of protease-resistant protein (PrP), which is encoded by a cellular gene of the host. This feature distinguishes prions sharply from virions. To date, no prion-specific nucleic acid has been identified which is required for transmission of disease.
Virus-like tubulofilamentous particles 23-26 nm in diameter have been seen consistently in the brains of all known spongiform encephalopathies. These particles have been investigated by H. K. Narang, Intervirology 32, 185-192 (1991) and H. K. Narang, D. M. Asher and D. C. Gajdusek, Proc. Natl. Acad. Sci. USA 85, 3575-3579 (1988) and 84, 7730-7734 (1987). They have a core of prions in a rod-like form. The prion rods are also termed scrapie-associated fibrils (SAF). Over the core is a layer of DNA, removable by DNAse and above that lies an outer protein coat which is digestible by a protease. In the Intervirology paper it is noted that no evidence as yet supports the existence of scrapie-specific nucleic acid, but it will be important to purify tubulofilamentous particles in order to characterise the nucleic acid and determine its relationship to the disease. Very recently H. K. Narang, N. S. Millar, D. M. Asher and D. C. Gajdusek, Intervirology 32, 316-324 (1991), have reported an increase in multimeric mitochondrial DNA in the brain of scrapie-infected hamsters, but, again, stated that there was no evidence for a scrapie-specific nucleic acid.
In their review entitled "The Search for Scrapie Agent Nucleic Acid", J. M. Aiken and R. F. Marsh, Microbiol. Rev. 54 (3) 242-246 (1990), concluded that the lack of success in identifying a scrapie-specific nucleic acid suggests that if there is such a nucleic acid, it would appear to be a very rare, or very small RNA or an RNA or DNA species having significant sequence similarity to nucleic acids present in uninfected tissue. Similarly, N. Meyer, V. Rosenbaum, B. Schmidt, K. Gilles, C. Miranda, D. Groth, S. B. Prusiner and D. Riesner, "Search for a putative scrapie genome in purified prion fractions reveals a paucity of nucleic acids", J. Gen. Virol. 72, 37-49 (1991) noted that prions resist inactivation by harsh procedures that specifically hydrolyse or modify nucleic acids, a feature which argues that they are probably devoid of polynucleotides, but that such negative results are always vulnerable to the criticism that a putative scrapie-specific nucleic acid might be so well protected by some unusual structure that its presence is yet to be detected. These authors concluded that the nucleic acid would have to be very small (of size less than 100 nucleotides), very efficient or heterogeneous in size.
The most reliable method of detecting an encephalopathy is histologically, especially by electron microscopy. See H. K. Narang and R. H. Perry, The Lancet, 335, 663-664 (March 17, 1990), and H. K. Narang, D. M. Asher and D. C. Gajdusek, Proc. Natl. Acad. Sci. USA 84, 7730-7734 (1987), but this requires brain tissue and is a slow and painstaking method.
It would be desirable to have a method of diagnosis based on nucleic acid identification. Such methods have been suggested. See H. Sorum, B. Hyllseth, R. Krona and O. Olsvik, Abstracts of the Annual Meeting of the American Society of Microbiologists 1988, page 363, Abstract No. C-188, who used a probe of DNA derived from the gene sequence coding for prion protein. Since, however, it is well known that prion protein is encoded by a normal chromosomal gene found in all mammals, including those affected by encephalopathies, the above-cited work has not gained acceptance. PCT Patent Application Publication No. WO89/11545 (Institute for Animal Health Ltd.) purports to describe a method of detection of scrapie by use of a restriction fragment length polymorphism (RFLP) linked to the so-called Sinc gene associated with short incubation times of sheep infected by scrapie. The RFLP is said to be located in a non-coding portion associated with the gene for prions. At best, this method would detect only sheep with the short incubation time characteristic. Hitherto, methods of diagnosis based on nucleic acid identification have not been successful or even likely to be successful, since an encephalopathy specific nucleic acid has eluded detection despite numerous attempts.
Several press articles in 1991 in the English newspaper "The Guardian" (27 April, 22 June, 16 August) report that the present inventor believes that he has found the genetic fingerprint for BSE and related diseases. No further information about the nature of the genetic fingerprint is given.